1. Field of the Invention
The present invention relates to measurement of plural target substances by use of a diamond electrode, and more particularly to a method of measuring concentrations of plural target substances in a sample from a change in a response electric current obtained by changing a potential in the interface of a diamond electrode. The present invention also relates to a concentration sensor to be used for that purpose.
2. Prior Art
Concentration sensors that electrochemically measure concentrations of various substances such as ions, inorganic compounds, organic compounds, polymeric compounds, living body-related substances etc. have been known. Such concentration sensors use an electrode for detecting changes in electric currents or potentials occurring in the interface of the electrode. Carbon-based materials, metal oxides, metals, semiconductors etc. are used as electrode materials in the electrodes in such concentration sensors. Sensor performance depends on the characteristics of the electrode materials used therein.
One of the prior art concentration sensors uses a platinum electrode. This sensor measures the oxidation current of hydrogen peroxide occurring on the platinum electrode to electrochemically determine the concentration of hydrogen peroxide ("Electrochemical Measuring Method, Volume 1", written by Fujishima, et al., and published by Gihodo Shuppan K.K., Nov. 15, 1984, pp. 238-239).
Another type prior art concentration sensor employs an electrode material for measuring the hydrogen peroxide concentration, and a molecule-recognizing substance such as enzyme, microorganism etc. Such concentration sensor is referred to by a biosensor, more specifically by a glucose sensor for use in measuring the blood sugar level or urine sugar level in blood or urine ("Advanced Sensor Handbook" written by Takahashi, et al., and published by K.K. Bifukan on May 20, 1994, pp. 335-339).
It is to be noted that each of these prior art concentration sensors aims at measuring a concentration of a single target substance and is not capable of measuring concentrations of plural target substance at the same time. Accordingly, the concentration of each target substance cannot be determined with one sensor where plural target substances are present in a system.
Further, in accordance with the prior art concentration measuring sensors and methods, a target substance would be influenced by other substances, resulting in a substantial decrease of accuracy of measurement. For example, the above-mentioned prior art hydrogen peroxide sensors determine hydrogen peroxide concentrations from oxidation current values of hydrogen peroxide when potentials of about +0.9 V (i.e. potential toward a reversible hydrogen electrode; every potential hereinafter described is referred to as a value toward a reversible hydrogen electrode) are applied to platinum electrodes. However, organic acids such as ascorbic acid, uric acid etc. would be oxidized at potentials of about +0.9 V, as well as hydrogen peroxide, so that the resulting oxidation current includes the oxidation currents of not only hydrogen peroxide but also of such organic acids. Accordingly, when this prior art concentration sensor is used for measuring hydrogen peroxide in a system containing organic acids such as ascorbic acid and uric acid, there would be a significant error in measurement.
For the same reason, there is a similar problem where blood glucose is measured by the above-described glucose sensor. When the glucose sensor is used, the following reaction (A) proceeds with a glucose oxidase catalyst: EQU Glucose+oxygen.fwdarw.glucolactone+hydrogen peroxide (A)
The amount of generated hydrogen peroxide is determined, and from the equivalent ratio, the glucose concentration is determined. Accordingly, the measurement of glucose in samples such as blood, urine etc. with the prior art glucose sensor is greatly influenced by ascorbic acid and uric acid present in blood or urine, so glucose concentrations cannot be accurately determined., similar to the above-described cases where hydrogen peroxide concentrations are determined in the system containing organic acids such as ascorbic acid, uric acid etc.
Further, molecular-recognizing substances such as glucose oxidase used in the glucose sensor are usually immobilized onto a film of substrates such as polymeric molecules etc. However, this has a poor stability and therefore could not used for a long time because of the elution of molecular-recognizing substances from the substrate, and reduction in the catalytic activity of the substances themselves.
Sensors using diamond as the electrode have also been known. For example, Japanese Patent Publication No. 2-22900 discloses an electrochemical test/analytical electrode consisting of diamond to which electrical conductivity has been added by ion injection. This publication also describes that such diamond electrode is favorably used as an electrochemical test/analytical indicator electrode because it has a wide potential region (potential window) free of hydrogen generation and oxygen generation (or metal elution) caused by electrolysis, and a low residual current (base current regarded as noise) in the potential window.
In Japanese Patent Laid-Open Publication No. 2-266253, it is disclosed that a conductive diamond layer, with impurities, is etched on a substrate by a gas phase method to produce an electrode, which is then coated with an enzyme-containing conductive resin to produce an enzyme sensor.
Japanese Patent Laid-Open Publication No. 8-240555 discloses a diamond film biosensor comprising a molecule-recognizing living body-related substance coated or immobilized on the surface of a transducer of a semiconductor diamond film.
Either of the sensors in the above-referenced two Japanese Patent Laid-Open Publications have a layer of a molecule-recognizing substance such as enzyme, microorganism etc. In fact, such molecule-recognizing substance layer adversely affects anti-corrosion and durability inherent in diamond itself. Further, each sensor merely measures a single substance. It is not at all suggested therein that concentrations of plural substances may be measured with a single sensor where there are plural substances in a system.
Studies on diamond electrodes have been rapidly increasing for the last few years. Among them, Swain et al. have used a diamond electrode to examine oxidoreduction characteristics of Fe(CN)6.sup.3-/4-, Ru(NH3)6.sup.3+/2+, IrCl6.sup.2-/3-, 4-methylcathecol, dopamine, methyl viologen, ferrocene, hydroquinone, ascorbic acid etc., and made a conclusion that the diamond electrode is promising as a sensor [G. M. Swain et al., Anal. Chem., 67, (1995), 2812-2821; G. M. Swain et al., Electrochem. Soc. Proceedings, 96-9, (1996), 138-148]. In their report, however, a response is observed where a single substance is present in a system. No measurement of plural substances in a multi-component system is suggested therein.
Zhu et al. show the relationship between hydrogen peroxide concentrations and response electric currents when diamond electrodes were used [J. Z. Zhu et al., Fresenius J. Anal. Chem., 352, (1995), 389-392], but measurement of plural substances in a multi-component system is not suggested.
Measurement of a specific substance in a multi-component system by using an electrode other than the diamond electrode has been reported by Kita et al. They conducted-electrolytic oxidation of glucose by modifying the surface of a platinum electrode with a Nafion membrane [H. Kita et al., J. Electroanal. Chem., 382, (1995), 103-110]. They reported that glucose could be measured solely under no influence of ascorbic acid etc. by using Nafion membrane. However, the target substance measured by this method is glucose only. Other substances, such as ascorbic acid, can not be measured. Because the platinum electrode has a narrow potential window, measurable substances in this method must be limited. Accordingly, this method is not applicable to measurement of concentrations of plural target substances in a multi-component system. In addition, Nafion membrane has a poor durability.
Polarography having a mercury electrode has been used for measurement of a specific substance in a multi-component system, by utilizing superior charateristics of the mercury electrode, that is, a high hydrogen overvoltage and an extremely large potential window at the reduction region ("Electrochemical Measuring Method, Volume 1", written by Fujishima, et al., and published by Gihodo Shuppan K.K., Nov. 15, 1984, pp. 197-203). However, mercury is dissolved in an oxidization area so that application of polarography using the mercury electrode is limited to a reduction area and, therefore, measurable substances are limited. Further, unlike the conductive diamond electrode proposed in the present invention, selective measurement of substances by use of a difference in reaction potential cannot be conducted.